DNA repair and recombination are required by organisms to prevent the accumulation of mutations and to maintain the integrity of genetic information. Compromised genetic material may result in cell cycle arrest, programmed cell death, chromosome loss or cell senescence. Alternatively, compromised genetic information may result in dysregulation of the cell cycle ultimately leading to increased cellular growth and tumor formation.
The repair of double-strand breaks (DSB) in DNA is an essential cellular process. DSB repair may occur during general cellular functions such as DNA repair (Friedberg et al., 1995, DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D.C.). In bacteria and yeast cells, DSB are predominately repaired by a homologous recombination pathway (Krasin and Hutchinson, 1977, J. Mol. Biol. 116:81-98; Mortimer, 1958, Radiat. Res. 9:312-16. In the budding yeast Saccharomyces cerevisiae the RAD52 epistasis group (Rad.sub.50 to Rad57, Mre11 and Xrs2) was identified in cells sensitive to ionizing radiation (reviewed in Friedberg, 1995; Petes et al., 1991, Recombination in yeast., p. 407-521. In J. R. P. J. R. Broach, and E. W. Jones (ed.), The Molecular and Cellular Biology of the Yeast Saccharomyces. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Later, some of the members of this group were shown to be important for recombinational repair (e.g., Rad51, Rad52, Rad54, Rad55, Rad57 (Malkova et al., 1996, Proc. Natl. Acad. Sci. USA 93:7131-36, Sugawara et al., 1995, Nature 373:84-86).
Among the members of the RAD52 epistasis group, ScRad51 is particularly interesting because it shares similarity with the Escherichia coli recombination protein, RecA. ScRad51 and RecA polymerize on double-stranded and single-stranded DNA (dsDNA, ssDNA) to produce a helical filament, and both enzymes catalyze an ATP-dependent strand exchange between homologous DNA molecules (Ogawa et al., 1993, Science 259:1896-99; Sung, 1994, Science 265:1241-4364; Sung and Robberson, 1995, Cell 82:453-61). ScRad51 and RecA share 30% homology over a span of about 220 amino acids, and each protein contains two conserved ATP binding motifs (Aboussekhra et al., 1992, Mol. and Cell. Biol. 12:3224-34; Basile et al., 1992, Mol. Cell. Biol. 12:3235-46; Sugawara et al., 1995, Nature 373:84-86).
ScRad51 repairs DSB by homologous recombination. DSB accumulate at recombination hot spots during meiosis in cells that lack ScRad51 (Sugawara, 1995), and ScRad51 localizes to meiotic nuclei (Bishop, 1994, Cell 79:1081-92) and promotes meiotic chromosome synapsis (Rockmill et al., 1995, Genes & Develop. 9:2684-95). Accordingly, it is thought that ScRad51 mediates meiotic recombination by binding to single-strands generated at DSB which are in strand pairing and exchange during meiosis (Sung and Robberson, 1995, Cell 82:453-61).
Direct and indirect protein--protein interactions are essential for RecA and ScRad51 function. The crystal structure of RecA suggests that a portion of the N-terminal region is involved in polymer formation (Story et al., 1993, Science 259:1892-96; Story et al., 1992, Nature 355:318-324) which was supported by genetic analysis that showed C-terminal truncations dominantly interfered with DNA repair in wild-type bacteria (Horii et al., 1992, J. Mol. Biol. 223:104-114; Tateishi et al., 1992, J. Mol. Biol. 223:115-129; Yarranton et al., 1982, Mol. Gen. Genet. 185:99-104). A similar self-association region occurs in the N-terminal region of ScRad51 and is essential for DNA repair (Donovan et al., 1994, Genes & Develop. 8:2552-2562; Shinohara et al., 1992, Cell 69:457-70). ScRad51 also associates with Rad52 and Rad55 (Hays et al., 1995, Proc. Natl. Acad. Sci USA 92:6925-6929; Johnson and Symington, 1995, Molec. Cell. Biol. 15(9):4843-4850; Milne and Weaver, 1993, Genes & Develop. 7:1755-1765) as well as other proteins. Other protein interactions may be inferred because a rad51 rad52A strain of S. cerevisiae was only partially complemented by Rad51 and Rad52 from Kluyveromyeces lactis (Donovan et al., 1994, Genes & Develop. 8:2552-2562), and because ScRad51 colocalized with Dmc1 to the synaptonemal complex (Bishop, 1994, Cell 79:1081-92). These data suggest that a large protein complex is necessary for recombinational repair and that disruption of any of the proteins in this complex hinders the repair of DSB.
RecA/ScRad51 homologues have been discovered in a wide range of organisms including the fission yeast Schizosaccharomyces pombe (Jang et al., 1994, Gene 142:207-11; Muris et al., 1993, Nuc. Acids Res. 21:4586-91; Shinohara et al., 1993, Nature Genet. 4:239-4358), lilies (Terasawa et al., 1995, Genes & Develop. 9:925-34), chickens (Bezzubova et al., 1993, Nucl. Acids Res. 21:1577-80), mice (Morita et al., 1993, Proc. Natl. Acad. Sci USA 90:6577-80; Shinohara et al. 1993, Nature Genet. 4:239-43) and humans (Shinohara et al. 1993; Yoshimura et al., 1993, Nucl. Acids Res. 21:1665), and appear to be involved in DNA repair and recombination based on the following evidence: 1) Conserved RecA homology--MmRad51 is 83% homologous, 69% identical to ScRad51, and 51% homologous, 28% identical to RecA. Shared homology between mammalian and yeast Rad51 suggest conserved function due to the remarkable similarity between other mammalian and yeast DNA repair pathways (reviewed in Cleaver, 1994, Cell 76:1-4); 2) Expression pattern--MmRAD51 is highly expressed in tissues involved in meiotic recombination such as testes (Morita et al., 1993, Proc. Natl. Acad. Sci USA 90:6577-80) and ovaries (Shinohara et al., 1993, Nature Genet. 4:239-43). Additionally, expression of the S. pombe MmRad51 homologue SpRAD51 increased after cells were treated with methyl methanesulfonate which provides further evidence of a DNA repair function (Jang et al., 1994, Gene 142:207-11); 3) Protein cellular localization--Mouse, chicken, and lily Rad51 localizes at discrete foci on meiotic chromosomes at varying concentrations during prophase 1, possibly on the lateral elements and recombination nodules, which suggests a role in the repair of DSB during meiotic recombination (Ashley et al., 1995, Chromosoma 104:19-28; Haaf et al., 1995, Proc. Natl. Acad. Sci. USA 92:2298-2302; Terasawa et al., 1995). Moreover, increasing concentrations of human Rad51, HsRad51, localize to the nucleus after exposure to DNA damaging agents which also suggests a repair function (Terasawa et al., 1995); 4) Filament formation on DNA--HsRad51 bind to ssDNA which demonstrates a potential for strand exchange (Benson et al., 1994, EMBO 13:5764-71); 5) Mouse cells with a rad51 mutation, designated rad51.sup.M1, displayed features that are known to be characteristic of unrepaired DSB in yeast cells (Lim and Hasty, 1996, In press) which include reduced proliferation, hypersensitivity to .gamma.-radiation, chromosome loss and programmed cell death.